Cycloartanes from Euphorbia aellenii Rech. f. and their Antiproliferative Activity.

The cytotoxic chloroform fraction of Euphorbia aellenii afforded two cycloartane type triterpenes-cycloart-25-en-3β,24-diol (1) and 24-methylene-cycloartan-3β-ol (2)-for the first time from this plant. Preparation of cycloartane derivatives, 3β, 24-O-diacetyl-cycloart-25-en as compound 3 and 3β-O-acetyl-24-methylene-cycloartan (4) were conducted by acetylating of 1 and 2, respectively. The structures of the isolated compounds were elucidated by spectroscopic methods and their activities evaluated by proliferation assay on human peripheral blood lymphocytes (PBLs). Comparing the results suggested that anti-proliferation effect of these compounds on PBLs might be due to the presence of free 3-OH group while masking the free OH groups by acetylation, could induce proliferation activity.


Introduction
Cycloartanes (9, 19-Cyclolanostanes) are one of the main tetra-cyclic triterpene skeletons including the side chain and characteristic cyclopropane ring (1). They are the major key intermediates in the phytosterols biosynthesis (1). In addition, these compounds are used as specific chemotaxonomic markers in Euphorbia genus, which comprises well over 2000 species in tropical and temperate zones of asia and other parts of the world. In iran, 70 species are reported, 17 of which are endemic (2). Within our recent study on triterpenoids, present work describes the isolation and structure elucidation of two cycloartane type triterpenes from Euphorbia aellenii Rech. f. (Figure 1) along with their anti-proliferative activity on human peripheral lymphocytes and their structurefunction studies on protein kinase C (PKC) that plays an important role, as an early event, in T-cell activation (3).

Plant material
The aerial flowering parts of Euphorbia aellenii Rich. F. (euphorbiaceae) were collected from populations growing in Galil-e-Shirvan (Iran) and identified by Dr. Yasamin Naseh, herbaceous sciences research center at ferdowsi university of mashhad. A herbarium specimen No. 2024 is preserved in the herbarium of the faculty of pharmacy, isfahan university of medical sciences (Iran).

Preparation of cycloartane derivatives
A mixture of cycloartane type compound (10 mg), acetic anhydride (1 mL) and pyridine (0.5 mL) were stirred at room temperature for three days. The final point of reaction was controlled by TLC and the reaction mixture was poured over ice to decompose the remaining acetic anhydride. Thereafter, suspension was extracted twice with describes the isolation and structure elucidation of two cycloartane type triterpenes from Euphorbia aellenii Rech. f. (Figure 1) along with their anti-proliferative activity on human peripheral lymphocytes and their structure-function studies on protein kinase C (PKC) that plays an important role, as an early event, in T-cell activation (3).  ethyl acetate (2:1 v/v), and evaporated in-vacuo to yield acetylated products (3 and 4), which was confirmed by Mass and H-NMR spectra (5).
Brine shrimp (artemia salina) cytotoxicity Shrimp eggs were added to a specific tank containing artificial seawater, hatched within two days and transferred to sample vials (1000, 100 and 10 μg/mL). 24 h later, keeping vials under illumination, the surviving shrimps were counted to obtain LC 50 and 95% confident intervals (4).

Proliferation assay
Peripheral human blood lymphocytes were incubated with different concentrations of the test compounds (0.5, 5, and 50 µg/mL (in triplicates) in supplemented RPMI-1640 along with phytohemagglutinin (PHA) at 37 º C in CO 2 environment for 72 h. Further incubation for 18 h after the addition of thymidine [ 3 H] (Amersham, UK) was done and cells were harvested using cell harvester (Innotech Dottikon, Switzerland). Finally, proliferation level was determined by the radioactivity count as CPM recorded from the Beta-scintillation counter (Beckman coulter, LS 6500, Fullerton, CA, USA) (6).

Docking methodology
Using the crystal structure of protein kinase C (PKC), retrieved from the protein data bank (PDB code: 1zrz), compounds (1-4) were selected for docking process and optimized by Polak-Ribiere conjugate gradient algorithm and AM1 semi empirical method, implemented in HyperChem. The optimized structures were used as the input of auto dock tools and the partial charges of atoms were calculated using gasteiger-marsili procedure (7). Merging nonpolar hydrogens, rotatable bonds were assigned; after removing the heteroatom including water molecules from protein, all missing hydrogens were added. Thereafter, determining Kollman united atom charges (8), non-polar hydrogens were merged to their corresponding carbons and as a final processing part in protein preparation, desolvation parameters were assigned to each atom. Using auto Grid tool, the grid maps (one for each atom type in the ligand, and one for electrostatic interactions) were constructed adequately large to include the active site of protein as well as significant regions of the surrounding surface. In all the cases, a grid map of 60 points in each Cartesian direction apart from a grid-point spacing of 0.375 A° (a quarter of the carbon-carbon single bond) were generated. By the ligand location in the complex, the maps were centered on the ligand's binding site, searching favorable interactions with the functional groups. Based on lamarckian genetic algorithm (9), using the pseudo-solis and wets local search method (10), Auto Dock Tools were employed to produce both grid and docking parameter files i.e. .gpf and .dpf files. Applying 2.0 A° clustering tolerance to construct clusters of the closest compounds, the initial coordinates of the ligand were used as the reference structure. In addition, for the internal validation phase, ligand structure (corresponding HETATM and CONECT records) was extracted from the pdb file of CDK 2 (2BTS). Later, assigning bond orders, missed hydrogens were added and a short minimization (100 steepest descent steps were taken, using MM + force field with a gradient convergence value of 0.05 Kcal/mol A°) to release any internal strain. At last, docking results (PKC-ligand complexes) were visualized using VMD1.8.6 (11).

Statistical analysis
The IC 50 values were calculated using Excel based program and reported as mean ± SD of the mean. Significance was attributed to p-values (p < 0.05) and the probability values obtained by the student t-test between the sample and control data.

Results and Discussion
Compound 1, white crystals with mp 180-184°C, showed the molecular formula of C 30 H 50 O 2 based on positive EI-HR-MS m/z 442.3784 (calc. for C 30 H 50 O 2 : 442.3811, ∆ 6.10 ppm) ), in accordance with the number and the multiplicity of 13 C-NMR spectra ( Table 1) , with reference to its axial and α-orientation, assigned the hydroxyl group as 3β-OH. Using HMBCs, the downfield carbinolic proton at δ H 3.99 (t, J = 6.3 Hz, H 24 ), showed connectivity with a pair of olefinic protons at δ H 4.80 (d, J = 1.2 Hz) and 4.90 br-s (each one H), suggesting a terminal methylene. As a whole, the six-degree of unsaturation and the 13 C-NMR data ( ] + , fragmented due to the elimination of parts of side chain (during a Mc Lafferty process), inferred presence of hydroxyl group in side-chain as is shown in Figure 2 (11). Regarding to these findings and the published data (11), compound 1 was identified as cycloart-25-en-3β,24-diol.

Proliferation assay
The anti-proliferation effect of the test compounds was determined by measuring the PHA-induced T-cell proliferation by determining radioactive thymidine incorporation. Comparison of pasitive, negative controls were included to assiss the activity of test compounds. cycloart-25-ene-3β, 24-diol (1) showed dose-dependent decrease in lymphocyte proliferation with IC 50 : 12.1 ± 0.6 µg/mL. This result was in conformity with another study by  (4), T-cells were stimulated by phytohemagglutinin (PHA) in the presence of three different concentrations of compounds. Significance differences between the means of compounds as compared to the control (PHA +ve) were calculated by using one-way ANOVA at p = < 0.05. (14) which showed cytotoxic activity against Ehrlich ascites tumor cells in mice. Likewise, 24-methylene-cycloartan-3βol (2), presented dose dependent inhibitory effect with IC50: 10.4 ± 0.1 µg/mL agreed with other published data supporting pain-relieving activity, and anti-inflammatory effect by TPAinduced ear oedema in mice (15). Masking free OH groups of 1 and 2 by acetylation, antiproliferative effect decreased significantly (IC 50 > 50 µg/mL). On the other hand, in the case of 3 with two acetyloxy groups (3-OAc and 24-OAc), proliferation of PBLs increased by 23-25% at the low concentration (0.5 µg/mL) in comparison with PHA (5 µg/mL) as positive control. However of the higher concentration 50 and 5 μg mL− 1 of 66-70% and 36-39% increas in prolification were absorb. These results suggested that the proliferation stimulatory activity on PBLs is related to the presence of 24-OAc function while anti-proliferation effect induced by free 3-OH group (Figure 3).

Docking results
In the internal validation phase of docking, bis(indolyl) maleimide was docked onto the PKC and the lowest energy pose for docking is shown in Figure 4. Superimposing the experimental and predicted conformations, the RMSD was achieved as 1.02 A°, considered as a successful docking (16) of such ligands with PKC. Thereafter, the proposed mechanism of the action was validated by docking the compounds (1-4) in the binding site ( Figure 5). All of four compounds tended to accept similar orientations, and docked into the active site of PKC. In compounds (1-2) forming hydrogenbond interactions between 3-OH and Pro-532, could explain the antiproliferative effect of T-cell derived PKC in-vitro. Therefore, based on this structure-function study, the presence of 3-OH could be correlated with the ability to deactivate PKC and inhibited T-cell proliferation. Likewise, hydrogen bond interaction between 24-O-acetyl group (3) and Asp-330 of PKC active site could be responsible for induction of lymphocyte proliferation derived PKC in-vitro.

Conclusion
For the first time Cycloart-25-en-3β,24-diol (in high quantity) and 24-methylene-cycloartan-3β-ol could be isolated from Euphorbia aellenii. Their immunomodulatory effects suggested that the proliferation stimulatory activity on PBLs is related to the presence of 24-OAc function while anti-proliferation effect induced by free 3-OH group. The SAR studies on PKC confirmed these results and provided further support for the role of PKC in transduction of activation signals in T-cells.
Masking free OH groups of 1 and 2 by acetylation, anti-proliferative effect decreased significantly (IC50 > 50 µg/mL). On the other hand, in the case of 3 with two acetyloxy groups (3-OAc and 24-OAc), proliferation of PBLs increased by 23-25% at the low concentration (0.5 µg/mL) in comparison with PHA (5 µg/mL) as positive control. These results suggested that the proliferation stimulatory activity on PBLs is related to the presence of 24-OAc function while anti-proliferation effect induced by free 3-OH group (Figure 3).   study, the presence of 3-OH could be correlated with the ability to deactivate PKC and inhibited T-cell proliferation. Likewise, hydrogen bond interaction between 24-O-acetyl group (3) and Asp-330 of PKC active site could be responsible for induction of lymphocyte proliferation derived PKC in-vitro.

Conclusion
For the first time Cycloart-25-en-3β,24-diol (in high quantity) and 24-methylene-cycloartan-3β-ol could be isolated from Euphorbia aellenii. Their immunomodulatory effects suggested that the proliferation stimulatory activity on PBLs is related to the presence of 24-OAc function while anti-proliferation effect induced by free 3-OH group. The SAR studies on PKC confirmed these results and provided further support for the role of PKC in transduction of activation signals in T-cells.